Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins

Abstract

Background: Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry.

Methods: A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, -2 and -3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, -2 and -3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs.

Results: 6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and -3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17% respectively. Phoxim also inhibited rDeg-2-GST (14.8%) and rDeg-GST-3 (20.6%) activities.

Conclusions: Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

Fig. 1

Conservation of glutathione S-transferase (GST) mu class elements and protein signature motifs in the N-terminal region of two GSTs derived from Dermanyssus gallinae. The N-terminal regions of two D. gallinae GST proteins (Deg-GST1 and Deg-GST-3) were aligned using the ClustalX algorithm with their two closest BLASTp matches: Metaseiulus occidentalis (XP_003742682.1 and XP_003747409.1), Nematostella vectensis (XP_001634653.1) and Haemaphysalis longicornis (AAQ74441.1). The signature motifs of mu class GSTs are annotated as follows: the conserved tyrosine active site residue (black shading), the four mu class elements (PR01267, bold and underlined) and the mu-loop structure (grey shading). The amino acid insertions conserved in Deg-GST-1 and XP_003747409.1 are in bold

Fig. 2

Phylogentic analysis of three glutathione S-transferase (Deg-GST) protein sequences derived from Dermanyssus gallinae. Three Deg-GST protein sequences (Deg-GST −1, −2 and −3) were aligned using the ClustalX algorthim along with a selection of homologous and protypal proteins belonging to the selected classes of GSTs: alpha, mu, beta, zeta, delta, omega epsilon and theta. The constituent proteins were manually truncated at the termini to eliminate regions of poor alignment. The phylogenetic tree was determined using the default parameters of MrBayes Baysian tree method with the Whelan & Goldman model (WAG) amino acid substitution model. The accession number and species of each protein is presented. The 3 Deg-GSTs are boxed. The clade credibility of each partition was assessed using posterior probabilities and is presented at each node

Fig. 3

Purity of recombinant versions of three Dermanyssus gallinae GST proteins (rDeg-GST −1, −2 and −3). rDeg-GST-1 (lane A), rDeg-GST-2 (lane B) and rDeg-GST-3 (lane C) were produced as fusion proteins with N-terminal His-tags and SUMO peptides then affinity purified through a HisTRAP column(GE healthcare) and the imidazole removed by dialysis against 10 mM Tris, 0.5 M NaCl, pH 7.4. Four μg of each rDeg-GSTs were denatured and electrophoresed on a 4–12 % Bis-Tris Novex gel (Invitrogen). Proteins were visualized with SimplyBlue™ SafeStain and molecular masses of the rDeg-GSTs estimated by comparison with SeeBlue® Plus2 pre-stained protein standards (Invitrogen)

Fig. 4

Enzymatic activity of three recombinant Dermanyssus gallinae GSTs proteins (rDeg-GSTs). The enzymatic activity of the purified rDeg-GST −1, −2 and −3 (panels a, b and c respectively) was determined over a 20 min period by measuring the increase in absorbance at 340 nm (A_340nm) resulting from the GST-driven processing of the colorimetric 1-chloro-2,4-dinitrobenzene (CDNB) substrate present at a range of concentrations (0 to 1 mM). All assays were performed in triplicate with a constant concentration of 2 mM reduced glutathionine (GSH). The A_340nm was adjusted for spontaneous substrate decay prior to calculating the mean specific activity (μmol/min/mg GST). The mean specific activity at the different CDNB concentrations is shown (± SEM, n = 3)